X-Band/Q-Band Spin-Trapping Development: Structure/Function Aspects of NOS-Generated Radicals
Balaraman Kalyanaraman, Professor and Chairman of Biophysics, and Director of the Free Radical Research Center
Joy Joseph, Associate Professor of Biophysics
The overall goal of this project is to use loop-gap resonators (LGRs) developed for X-band using 10 microliters of sample, which will significantly enhance our capability to detect superoxide radicals formed from nitric oxide synthases and from mitochondria. In order to routinely use the LGR/superoxide spin-trapping assay in cellular and biological systems, we need to synthesize and develop spin traps that are inherently stable and also form sufficiently stable spin adducts that exhibit distinct and easily identifiable EPR spectral patterns. Previously synthesized solid nitrone traps contain an ester moiety that is likely to undergo esterase-catalyzed intracellular metabolism. To circumvent this problem, we replace the ester group with an acrylamide group that still retains the electron-withdrawing ability of, vis-à-vis, 5-t-butylaminocarbonyl 5-methyl 1-pyrroline N-oxide (BAMPO). This cyclic amide trap is a water-soluble solid that is readily purified by recrystallization. The EPR spectrum of BAMPO-OOH is reasonably stable (half-life, 15 min) and exhibits a simple yet characteristic spectral pattern (aN = 13.2 G, aH = 12.4 G, and aH (gamma) = 1.3 G). The BMPO-OOH does not decay to the corresponding hydroxyl adduct. Thus, BAMPO appears to be a promising nitrone trap for detecting superoxide under conditions where hydrolysis of esters is favored. We are currently using BAMPO and other mitochondrially-targeted nitrones, in conjunction with an LGR, to detect superoxide formed from mitochondria in the presence of complex-1 inhibitors.